Project Summary The Broad Aim of this proposal is to advance the field of stem cell biology, cell-based diagnostics, and cell sourcing for cell therapy and drug development using a new pathway for systematic isolation of specific stem cell and progenitor cell subtypes from the plurality of niches in musculoskeletal connective tissues that we refer to as ?Performance-Based Selection? (PBS). Our goal is to provide patients, clinicians and researchers with the knowledge and rigorous and reproducible tools and standards that they need, to identify and obtain the best possible cells for use in regenerative and joint preservation therapies, drug discovery and basic research. Mesenchymal Stromal Cells (MSCs) derived from colony founding Connective Tissue Progenitors (CTPs) in bone and other tissues are widely valuable for cellular therapy. However, batch to batch MSC variation represents a profound point of ongoing ?pain? in the biomedical research community that is limiting the clinical evaluation and performance of MSC-based products. Clinical therapies demand high levels of repeatability and reproducibility. Minimizing variation is essential to advancing the emerging field of cellular therapies. We hypothesize that a large source of this variation is the failure of traditional methods of MSC fabrication based on ?Competitive Expansion? (CE), where the user simply puts all cells from tissue source into culture, where they compete. No choice is made regarding cells to include or exclude. The user simply accepts an outcome that may be predetermined by random variation in the type and mix of colony founding cells in the initial culture. This proposal integrates several unique technology platforms to enable rapid and effective progress in overcoming these limitations, specifically: 1) Standardized robotics for automated large-field-of-view (LFOV) (?Google Earth?) imaging and image analysis to identify and measure CTPs and their progeny. 2) Time Lapse Videomicroscopy that enables continuous tracking of the formation of each CTP-derived colony from the time of plating. Reversing the video enables us, for the first time, to see and to characterize colony founding CTPs as they exist immediately after removal from our bodies and before they divide. 3) An automated robotic platform (Cell X?) that enables both the selection (?Picking?) of individual CTP-derived clones as well as the automated processing that is needed to expand their progeny ?hands free?, enabling vastly greater precision and reproducibility than current manual methods. This proposal builds the knowledge that CTPs vary widely in biological potential. Using these tools, we will determine the attributes of colony founding CTPs that should or should not be included in MSC manufacturing and then provide the means to use this knowledge to achieve high quality and highly reproducible outcomes.